Of all the genes in a genome, only a small fraction are expressed in any individual cell. The temporal and spatial regulation in gene expression determines life processes. Many pathological developments, such as oncogenesis, are driven by gene expression. Identification of multiple genetic alterations as they occur in the genome is critical to understanding the molecular genetic events. Sequence analysis of nucleic acid molecules is essential to understanding and diagnosing disorders. Genetic alterations identified as being related to diseases can then be used as a guide for drug discovery. In the past two decades, the invention of techniques for immobilizing nucleic acid molecules on a solid phase has had a profound impact on the progress of the nucleic acid analysis, such as the use of DNA chips for gene expression analysis and solid phase primer extension for single nucleotide polymorphism analysis. Automation of solid phase oligonucleotide synthesis has helped propel the advancement of molecular biology, which undoubtedly has laid the foundation of the modern understanding of life sciences.
In general, the present invention features a method for producing an immobilized oligonucleotide attached to a substrate via a C-5xe2x80x2 position and having a terminal C-3xe2x80x2 position.
In one aspect, this invention is directed to a method of producing an immobilized oligonucleotide on a substrate to which a first nucleotide is covalently attached via its C-5xe2x80x2 oxygen. The first nucleotide can be a nucleotide monomer or the 5xe2x80x2 terminal nucleotide of a nucleotide polymer. In general, such a first nucleotide includes a modified nucleotide tethered to a support substrate through a linking group. In particular, the modified nucleotide is constructed such that the C-5xe2x80x2 end of the nucleotide is tetherable to the linking group and the C-3xe2x80x2 end is available for further controlled modification, e.g., addition of other nucleotides in specific sequences to the immobilized nucleotide. In the case of adding a nucleotide monomer as the first nucleotide, the C-3xe2x80x2 end is the C-3xe2x80x2 of the nucleotide monomer. In the case of adding a nucleotide polymer as the first nucleotide, the C-3xe2x80x2 end is the C-3xe2x80x2 of the terminal nucleotide of the polymer. Additionally, the linking group is of sufficient length to allow the immobilized nucleotide to be used to synthesize and screen arrays of nucleotide oligomers, e.g., enzymatic C-3xe2x80x2 primer extension.
In another aspect, the invention provides a method for in situ solid phase oligonucleotide synthesis with C-5xe2x80x2 attached to the substrate, thereby producing oligonucleotides which are a polymer of nucleotides. The method covers an in situ deprotection-activation-coupling cycle of oligonucleotide synthesis that includes covalently coupling a modified nucleotide via its C-5xe2x80x2 oxygen to an immobilized hydroxyl, wherein the modified nucleotide includes a C-3xe2x80x2 photolabile protecting group and a C-5xe2x80x2 hydroxyl group, and also wherein the immobilized hydroxyl group is activated with a phosphorous activating group. The synthesis includes sequentially deprotecting photolabile group from the C-3xe2x80x2 oxygen of an immobilized nucleotide at terminus, activating the C-3xe2x80x2 oxygen at terminus, in situ, with an activating phosphorous group, and coupling C-3xe2x80x2 protected nucleotides to the activated nucleotide at terminus. Optionally, the cycles of deprotecting, activating, and coupling can be repeated until a desired oligonucleotide is obtained. Typically, the immobilized C-3xe2x80x2 oxygen is activated with a phosphorous group such as a phosphoramidite, [(i-Pr)2N]POCH2CH2CN. The produced oligonucleotide can be further involved in enzyme-catalyzed reactions, e.g., polymerase mediated primer extension.
In further another aspect, the invention provides a method for an oligonucleotide synthesis in a direction of 5xe2x80x2 to 3xe2x80x2, thereby producing oligonucleotides that are a set of specific nucleotides. The method covers a deprotection-activation-coupling oligonucleotide synthesis which consists of a nucleotide or an oligonucleotide having a free terminal C-3xe2x80x2 hydroxyl and a terminal C-5xe2x80x2 that is blocked by a group, wherein the free terminal C-3xe2x80x2 hydroxyl is activated with a phosphorous activating group. The synthesis includes sequentially deprotecting a photolabile protecting group from the C-3xe2x80x2 oxygen of a nucleotide at terminus, activating the C-3xe2x80x2 oxygen, in situ, with an activating phosphorous group, and coupling another C-3xe2x80x2 photolabile protected nucleotides to the activated nucleotide at terminus. Optionally, the cycles of deprotecting, activating, and coupling can be repeated until a desired oligonucleotide is obtained. Typically, the C-3xe2x80x2 oxygen is activated with a phosphorous group such as a phosphoramidite, e.g., [(i-Pr)2N]POCH2CH2CN.
The invention also features an array. The array includes a substrate having a plurality of addressable sites. Each of the sites of the plurality has an oligonucleotide covalently attached to the substrate via its C-5xe2x80x2 oxygen atom. Each site of the plurality can be directly adjacent to at least one other site. The sequence of each oligonucleotide of a site can be unique among the plurality. Addressable sites other than the sites of the plurality can be disposed on the array. The array can have a density of addresses and oligonucleotides described below. A spatially selective irradiation technique can be used to make such an oligonucleotide array. Generally, the method covers an in situ deprotection-activation-coupling oligonucleotide synthesis to covalently couple the C-5xe2x80x2 position of a non-immobilized nucleotide or oligonucleotide to a substrate to form an immobilized oligonucleotide having a photolabile group protected C-3xe2x80x2 oxygen available for the attachment of subsequent C-3xe2x80x2 protected nucleotides. The substrate includes a plurality of immobilized nucleotide starters arranged on the substrate as a 2-dimensional array. The synthesis for covalently coupling the C-5xe2x80x2 position of a non-immobilized nucleotide or oligonucleotide to a substrate includes selectively removing photolabile groups from a subset of immobilized nucleotides or oligonucleotides in the array by irradiating the subset to produce an hydroxyl group at the C-3xe2x80x2 terminus. The C-3xe2x80x2 hydroxyl group on the immobilized nucleotide at terminus can be activated again in-situ to form phosphoramidite for coupling the next non-immobilized nucleotide or oligonucleotide having a C-5xe2x80x2 hydroxyl group. Alternatively, the C-3xe2x80x2 hydroxyl group on the immobilized nucleotide can couple with a non-immobilized nucleotide or oligonucleotide having an C-5xe2x80x2 activated group and a C-3xe2x80x2 photolabile protecting group. The sequence and the length of the immobilized oligonucleotide can be chosen for specific applications. Photolabile protecting groups include, but are not limited to, NVOC, MBNPEOC, and MeNPOC.
Within the scope of this invention is a method to synthesize a nucleotide that is activated at C-5xe2x80x2 and photolabile group-protected at C-3xe2x80x2. The method includes protecting the C-5xe2x80x2 hydroxyl group, attaching a photolabile protecting group to the C-3xe2x80x2 oxygen, deprotecting the C-5xe2x80x2 hydroxyl group, and attaching an activated phosphorous group to the C-5xe2x80x2 oxygen of a ribonucleic or deoxyribonucleic acid. Typically, the activated phosphorous group is phosphoramidite, i.e., [(i-Pr)2N]POCH2CH2CN. In general, the stereochemistry at C-1, i.e., where the base is attached to the sugar ring, can be selected to improve the synthetic yield of the C-5xe2x80x2 activated, C-3xe2x80x2 photolabile group protected nucleotide. Also contained within the scope of this invention is a method to covalently couple the C-5xe2x80x2 activated terminus of a monomeric nucleotide to a surface modified functional group on a substrate to form an immobilized nucleotide primer with an C-3xe2x80x2 oxygen available for the attachment of the next C-5xe2x80x2 activated, C-3xe2x80x2 protected nucleotide.
The invention features a C-3xe2x80x2 protected nucleotide having the formula 
wherein X is selected from the group consisting of a photolabile protecting group and a chemically labile protecting group; R is selected from the group consisting of hydrogen and hydroxyl; P is selected from the group consisting of hydrogen, a phosphorous activating group, and a phosphate or derivative thereof, and Base is selected from the group consisting of pyrimidine, purine, and derivatives thereof.
C-3xe2x80x2 protected nucleotide can have the formula 
wherein R1, R2, R3 each independently is selected from the group consisting of hydrogen, C1-C10 alkyl, C2-C10 alkenyl, aryl, benzyl, and C1-C10 alkoxyl; R4 is selected from the group consisting of C1-C10 alkyl, C2-C10 alkenyl, aryl, and benzyl; and P is selected from the group consisting of hydrogen and [(i-Pr)2N]POCH2CH2CN.
The C-3xe2x80x2 protected nucleotide can also have the formula 
wherein R1, R2, R3 each independently is selected from the group consisting of hydrogen, C1-C10 alkyl, C2-C10 alkenyl, aryl, benzyl, and C1-C10 alkoxyl; R4 is selected from the group consisting of C1-C10 alky, C2-C10 alkenyl, aryl, and benzyl; and P is selected from the group consisting of hydrogen and [(i-Pr)2N]POCH2CH2CN.
Embodiments of the aspects of the invention may include one or more of the following. The terminal position of the linking group for coupling with a C-3xe2x80x2 protected nucleotide is a functional group. The functional group is activated in-situ to form a phosphoramidite. The terminal phosphoramidite is coupled with a C-5xe2x80x2 hydroxyl group of a nucleotide bearing a photolabile group protecting the C-3xe2x80x2 oxygen. The immobilized monomeric nucleotide is covalently attached to the linking group via the C-5xe2x80x2 oxygen. Preferably, the nucleotide of the immobilized monomeric nucleotide is deoxyinosine.
The invention provides one or more of the following advantages. The in situ deprotection-activation-coupling oligonucleotide synthesis is economical and versatile and generates solid phase phosphoramidite that exhibits unexpected high efficiency in coupling with sequentially added C-3xe2x80x2 photolabile group protected nucleotides. The in situ deprotection-activation-coupling oligonucleotide synthesis provides high yield in-situ surface activation, e.g., substantially quantitative and stable, for forming surface phosphoramidite at the immobilized C-3xe2x80x2 position. The photolabile group-protected C-3xe2x80x2 nucleotides provide sufficient solubility in organic solvents for conducting nucleotide coupling reactions and have a short half-life that can improve the efficiency of photo-deprotection at the C-3xe2x80x2 position. Additionally, excess C-3xe2x80x2 photolabile group protected nucleotides can be recycled and directly used in the later coupling reactions, whereas coupling methods for coupling non-immobilized, phosphoramidite-containing nucleotides require a large excess of the phosphoramidite containing nucleotide (up to 50 folds) which subsequently decomposes in the coupling reaction. Inosine is known to help stabilize hybridization with another DNA molecule.
More than a single disease associated target gene can be tested to reveal more detailed disease information by producing DNA chips which contain a series of spatially arrayed specific immobilized nucleotide primers. Testing spatially arrayed immobilized nucleotide primers on DNA chips also will provide more precise disease diagnostic data of a patient and therefore more efficient treatment. Use of the PCR technique to identify the target genes with the primer-containing DNA chips is more reliable than hybridization alone. Currently, PCR-based disease diagnostics have not been widely used because of the cost concern. Thus, the immobilized oligonucleotides containing DNA chips of this invention would be more useful, accurate and relatively inexpensive.
Due to the orientation of the immobilized oligonucleotides, bound C-5xe2x80x2 and terminal C-3xe2x80x2, the immobilized oligomer acts as an oligonucleotide array for use in primer extensions in the presence of an enzyme, such as polymerase, DNA templates, and nucleotide triphosphates. The ability to perform primer extension in an array format has many important applications including single nucleotide polymorphism (SNP) analysis in pharmacogenomics, transcriptional profiling and on-chip gene sequencing. Unlike immobilized oligonucleotides having C-3xe2x80x2 bound and C-5xe2x80x2 at the terminal position which can only be used in hybridization for genetic analysis, the immobilized oligonucleotides having C-5xe2x80x2 bound and C-3xe2x80x2 at the terminal position can be used as primers for polymerase mediated primer extension. The enzymes used in the primer extension can also avoid mismatches during hybridization but before the enzyme mediated primer extension occurs to provide an advantage of proofreading the matched sequences of the DNA probes. The proofreading ability of the enzyme forms the basis of the detection of single nucleotide polymorphism (SNP).